Studies on the characterization of the anti-trypanosomal factor (ATF) produced by Pseudomonas fluorescens against the Tulahuen strain of Trypanosoma cruzi disclosed further the occurrence of two active small components, ATF II and ATF-III, which exhibited approximate molecular weights of 1,355 and 1,060 when chromatographed with Sephadex G-25. Gel filtration with Sephadex G-200 disclosed that the large active component previously described (ATF-I) exhibited a molecular weight higher than 400,000. The most active fraction was ATF-II, the least, ATF-I. In vitro studies with cultured fibroblasts revealed that the ATF-III produced significant alteration of flagellate populations and amastigote multiplication. Although ATF-II-induced lysis was produced in vivo, these observations indicated that the reduced parasitemia occurring in the ATF-II-treated mice and the control of the acute infection was associated also with an interruption of the parasitic cycle at the tissue level.